全 文 ：植物病理学报
ACTA PHYTOPATHOLOGICA SINICA　 41(1): 44-48(2011)
Received date: 2009-04-29; Revised date: 2010-09-08
Foundation item: National Natural Science Foundation of China (30200219; 30671684; 30500392)
Corresponding author: YE Jian-ren, professor, major in Forest Pathology; Tel: 025-85427773, E-mail: jrye@njfu. com. cn
Biography: TAN Jia-jin (1973 - ), male, native of Feidong, Anhui, associate professor, Ph. D. , major in Forest Patholgy and Plant Nema-
tology, E-mail: tanjiajin@tom. com.
Effects of pine wood nematode, Bursaphelenchus
xylophilus, on some physio-biochemical indexes
of Japanese black pine, Pinus thunbergii
TAN Jia-jin, YE Jian-ren, HAO De-jun, WU Xiao-qin, CHEN Feng-mao
(Jiangsu Key Laboratory for Prevention and Management of Invasive Species, College of Forest
Resources and Environment, Nanjing Forestry University, Nanjing 210037, China)
Abstract: Pine wilt caused by pine wood nematode, Bursaphelenchus xylophilus, is a serious disease for
pines. In this paper, changes of some physio-biochemical indexes in stem of 3-4 years old Japanese black
pine, Pinus thunbergii inoculated with B. xylophilus were studied. The results showed that after nematode in-
vasion, the soluble carbohydrate in pine seedlings decreased, and it was more significant at the middle and late
stages of disease development. The soluble protein and ascorbic acid in pine seedlings decreased gradually,
and the free amino acid in pine seedlings also decreased. It was suggested that the content of soluble protein in
stems of pine could be used for early diagnosis of the disease. Roles of the four physio-biochemical indexes in
pathogenic mechanism of the disease were discussed.
Key words: Bursaphelenchus xylophilus; Pinus thunbergii; ascorbic acid; free amino acid; soluble carbohy-
drate; soluble protein
松材线虫对黑松一些生理生化指标的影响　 谈家金, 叶建仁, 郝德君, 吴小芹, 陈凤毛　 (南京林
业大学森林资源与环境学院,江苏省有害生物入侵预防与控制重点实验室, 南京 210037)
摘要: 松材线虫病是松树的一种毁灭性病害。 本文对接种松材线虫后 3 ~ 4 年生黑松茎部的一些生理生化指标的变化进
行了研究。 结果显示,接种线虫后,松苗的可溶性糖含量降低,在病情发展中期和后期比早期降低更显著。 松苗的可溶性
蛋白质和抗坏血酸含量逐渐降低,游离氨基酸含量也降低。 松树茎部的可溶性蛋白质含量可用于病害的早期诊断。 此外,
对这 4 个生理生化指标在病害致病机理中的作用进行了讨论。
关键词: 松材线虫; 黑松; 抗坏血酸; 游离氨基酸; 可溶性糖; 可溶性蛋白质
中图分类号: S763. 1　 　 　 　 　 文献标识码: A　 　 　 　 　 文章编号: 0412-0914(2011)01-0044-05
　 　 Pine wilt caused by pine wood nematode, Bur-
saphelenchus xylophilus, is a serious disease of
pines. The pine wood nematode has spread into
many countries in Asia and Europe from North A-
merica and killed millions of pine trees in Japan and
China[1,2] . Presently, it makes a great threat to
those healthy forests all over the world.
Despite much research on the disease, the patho-
genic mechanism of the disease was still obscure,
which resulted in a great difficulty for disease con-
trol. Studies on physio-pathological changes in
pines, so as to find significant internal symptom and
　
　 1 期 TAN Jia-jin,et al.:Effects of pine wood nematode, Bursaphelenchus xylophilus , on some physio-biochemical indexes of Japanese black pine, Pinus thunbergii
acute index, will have an extensive application in
rapid quarantine, early diagnosis and screening for
disease-resistant pines. According to the former re-
search, some physio-biochemical indexes were pro-
posed for early diagnosis, and some resistance-rela-
ted physio-biochemical indexes were found[3 - 8] . In
this paper, changes of the contents of soluble carbo-
hydrate, soluble protein, resistance-related free ami-
no acid and ascorbic acid in stems of 3-4 years old
Japanese black pine, Pinus thunbergii inoculated
with pine wood nematode were studied.
1　 Materials and methods
1. 1　 Pine wood nematode and inoculation
Pine wood nematodes were extracted, cultured
and disinfected before being used for inoculation as
previously described[8] . Japanese black pines, 3-4
years old, were inoculated by the bark inoculation
method in a field of Nanjing Forestry University as
previously described[8] . A total of 15 pine seedlings
were inoculated, and another 15 pine seedlings were
injected with aseptic water as the control.
1. 2 　 Disease observation and nematode ex-
traction
External symptoms ( needle discolour and wil-
ting) of each pine seedling were observed every 5
days after inoculation. Pine wood nematodes were
extracted from a 20% subsample by weight of stem
samples 5, 10, 15, 20 and 70 days after inocula-
tion. Meanwhile, xylem resin exudation of the stem
samples was also observed.
1. 3 　 Determination of soluble carbohydrate
content of samples
Three inoculated and three control plants were
collected from the field 5, 10, 15, 20 and 70 days
after inoculation. Needles and roots were removed,
and the stems were cut into 0. 5 - 1. 0 cm-long
pieces and mixed. 0. 5 g of stem samples were used
for determination of soluble carbohydrate content by
anthrone method[9] . There were three replicates for
each treatment ( nematode inoculated seedlings and
seedlings injected with water ) . Comparison of
means was made for each treatment using t-test at the
5% level of significance.
1. 4 　 Determination of soluble protein con-
tent of samples
The method and sampling times were as de-
tailed above. 1. 0 g of stem samples were ground to
powder in liquid nitrogen, suspended in 5 mL of
0. 05 M oxalic acid -0. 2 mM EDTA solution, soni-
cated for 5 min, centrifuged for 10 min at 10 000
rpm after 0. 5 h. The supernatant was used for deter-
mination of soluble protein by Bradford method[10] .
There were three replicates for each treatment. Com-
parison of means was made for each treatment using
t-test at the 5% level of significance.
1. 5 　 Determination of free amino acid con-
tent of samples
The method and sampling times were as de-
tailed above. 0. 5 g of stem samples were used for
determination of free amino acid by triketohydrin-
dene hydrate method[11] . There were three replicates
for each treatment. Comparison of means was made
for each treatment using t-test at the 5% level of sig-
nificance.
1. 6 　 Determination of ascorbic acid content
of samples
The method and sampling times were as de-
tailed above. 1. 0 g of stem samples were pretreated
as described in determination of soluble protein con-
tent. The supernatant was used for determination of
ascorbic acid by the method as previously de-
scribed[9] . There were three replicates for each treat-
ment. Comparison of means was made for each
treatment using t-test at the 5% level of signifi-
cance.
2　 Results
2. 1　 Disease development in pine seedlings
After nematode invasion, exudation of xylem
54
　
植物病理学报 41 卷
resin was interrupted, then the needles wilted and
the plants died. The seedlings appeared normal 15
days after inoculation, while cessation of resin exu-
dation began to occur in the stem. At 25 days after
inoculation, the seedlings began to show external
symptoms, a few leaves on new season branches be-
gan to wilt. New leaves in all pine seedlings wilted,
and plants were dying or dead 70 days after inocula-
tion. The appearance of the seedlings and the stem
xylem were normal in all control pine seedlings.
Pine wood nematodes were found in all inoculated
plants, while not in control plants.
2. 2　 Change of soluble carbohydrate content
of samples
After nematode invasion, the soluble carbohy-
drate in stems of pine seedlings decreased, more sig-
nificantly at middle and late stage of disease deve-
lopment than that at early stage. At 5 days after in-
oculation, the content of soluble carbohydrate in in-
oculated plants was 31. 91 mg / g, decreased 24. 67%
compared to that in the control, and the values
reached 21. 14 and 12. 17 mg / g, decreased 54. 96%
and 68. 24% compared to that in the control 20 and
70 days respectively after inoculation (Fig. 1) . Du-
ring the whole experiment, the content of soluble
carbohydrate in control plants was almost constant.
Fig. 1 　 Change of soluble carbohydrate con-
tent in stems of Pinus thunbergii inocu-
lated with Bursaphelenchus xylophilus
Each value was mean of three replications; Bar is SE ( stan-
dard error) .
2. 3 　 Change of soluble protein content of
samples
After nematode invasion, the soluble protein in
stems of pine seedlings decreased gradually. With
the time elapsed after inoculation, the content of sol-
uble protein in inoculated plants decreased to 3. 07,
2. 66, 2. 24, 1. 96 and 0. 92 mg / g, respectively,
while that in control plants was almost constant
(Fig. 2) .
Fig. 2 　 Change of soluble protein content in
stems of Pinus thunbergii inoculated
with Bursaphelenchus xylophilus
Each value was mean of three replications; Bar is SE ( stan-
dard error) .
2. 4 　 Change of free amino acid content of
samples
The data showed that free amino acid in stems
of pine seedlings decreased after nematode invasion.
The content of amino nitrogen of free amino acid in
inoculated plants was 31. 31, 27. 13, 18. 36 and
13. 05 μg / g, while that in the control were 42. 73,
40. 26, 51. 73 and 28. 29 μg / g, respectively after
10, 15, 20 and 70 days of inoculation (Fig. 3) . At
20 days after inoculation, shortly before external
symptoms appeared, the amino nitrogen in inocula-
ted plants decreased most significantly compared to
the control.
64
　
　 1 期 TAN Jia-jin,et al.:Effects of pine wood nematode, Bursaphelenchus xylophilus , on some physio-biochemical indexes of Japanese black pine, Pinus thunbergii
Fig. 3　 Change of free amino acid content in
stems of Pinus thunbergii inoculated
with Bursaphelenchus xylophilus
Each value was mean of three replications; Bar is SE ( stan-
dard error) .
2. 5　 Change of ascorbic acid content of sam-
ples
After nematode invasion, the ascorbic acid in
stems of pine seedlings decreased gradually. The
contents of ascorbic acid in inoculated plants was
0. 91, 0. 80, 0. 75, 0. 53 and 0. 25 mg / g, decreased
5. 46% , 10. 64% , 24. 12% , 47. 78% and 74. 14% ,
respectively compared to that in the control with the
time elapsed after inoculation (Fig. 4) . During the
whole experiment, the content of ascorbic acid in
control plants was almost constant.
Fig. 4 　 Change of ascorbic acid content in
stems of Pinus thunbergii inoculated
with Bursaphelenchus xylophilus
Each value was mean of three replications; Bar is SE ( stan-
dard error) .
3　 Discussion
In this paper, the results showed that after nema-
tode invasion, the content of soluble carbohydrate in
pine seedlings decreased, more significantly at mid-
dle and late stage of disease development than that at
early stage. It was supposed that the disease de-
velopment of pine wilt could be divided into three
stages: early, middle and late stage. The early stage
is before resin exudation abnormity and the late stage
is after external symptoms appearance. Previous
study[12] showed that under water stress, the content
of soluble carbohydrate in plants always increased,
and it was an adaptive response of plants to water
stress. It was reported that water stress was an im-
portant pathological process of pine wilt[6] . Howe-
ver, a decrease of soluble carbohydrate content in in-
oculated pine seedlings suggested that a more impor-
tant pathological process might decide disease deve-
lopment caused by nematode invasion.
The data also showed that after nematode inva-
sion, the content of soluble protein in pine seedlings
decreased gradually. Soluble protein includes many
important physiological regulatory substances, such
as some pathogenesis-related proteins. Further ef-
forts need to be made to resolve whether nematode
invasion has dramatic effect on those proteins.
The data indicated that the free amino acid in
pine seedlings also decreased after nematode inva-
sion. The content of amino acid showed a lower le-
vel in some disease-resistant plants[13], Masson pine
(P. massoniana) with 9 kinds of low-content free
amino acids was resistant to pine wilt[14] . Decrease
of free amino acid in Japanese black pine after nema-
tode invasion might be a disease-resistant reaction,
and this reaction might reach the strongest before ex-
ternal symptoms appeared.
Ascorbic acid, also called vitamin C, is an im-
portant vitamin, exists extensively and is one of the
members of defensive system against free radical in-
jury in organisms[9] . The defensive system includes
enzymes and non-enzymes. Superoxide dismutase,
catalase, etc. are members of enzymic defensive
system, and ascorbic acid, reduced glutathione, etc.
74
　
植物病理学报 41 卷
are members of non-enzymic defensive system. It
was reported that activities of superoxide dismutase
and catalase decreased gradually in Japanese black
pine seedlings inoculated with pine wood nema-
tode[15] . In this study, the content of ascorbic acid
in pine seedlings also decreased gradually after nema-
tode invasion. The results suggested that decline of
the enzymic and non-enzymic defensive systems
might induce accumulation of free radicals, and ex-
cessive free radicals would injure the pine and finally
cause death of pines.
Early diagnosis is important in controlling pine
wilt. Characteristic physio-biochemical changes be-
fore resin exudation abnormity in pines could be
used for early diagnosis. In this study, it was found
that at 10 days after nematode inoculation, i. e. , be-
fore resin exudation was interrupted, the soluble pro-
tein in inoculated plants was 2. 66 mg / g, lower than
that in control plants during the whole experiment
(3. 30-3. 45 mg / g) . Thus, the content of soluble
protein in stems of pines could be used for early dia-
gnosis of the disease. However, the results were ob-
tained by experiments conducted on pine seedlings.
Further research was required to learn whether it
would be feasible for practical diagnosis.
Acknowledgement: We sincerely thank Dr. Yin
Tong-ming for his comments and revision on this
manuscript.
References
[1] 　 Fielding N J, Evans H F. The pine wood nematode
Bursaphelenchus xylophilus ( Steiner and Buhrer )
Nickle ( = B. lignicolus Mamiya and Kiyohara): an
assessment of the current position[J] . Forestry, 1996,
69(1): 35 -46.
[2] 　 Han Z M, Hong Y D, Zhao B G. A study on pathoge-
nicity of bacteria carried by pine wood nematodes.
Journal of Phytopathology, 2003, 151: 683 -689.
[3] 　 Guo D S, Zhao B G, Li Z Z. The pathogenic mecha-
nism of pine wilt disease caused by Bursaphelenchus
xylophilus ( in Chinese)[J] . Journal of Nanjing Fore-
stry University (南京林业大学学报), 2000, 24(4):
64 -68.
[4] 　 Tan J J, Wu H P, Yang R Z, et al. Effect of Bur-
saphelenchus xylophilus on several physiological and
biological indexes of Pinus thunbergii ( in Chinese)
[J] . Forest Pest and Disease (中国森林病虫),
2001, 20(4): 3 -5.
[5] 　 Tan J J, Feng Z X. Advance on physiopathology of
pine wood nematode disease ( in Chinese)[J] . Jiangxi
Science (江西科学), 2001, 19(1): 27 -30.
[6] 　 Yang B J. Advance in research of pathogenetic mecha-
nism of pine wood nematode ( in Chinese)[J] . Forest
Pest and Disease (中国森林病虫), 2002, 21(1): 27
-31.
[7] 　 Tan J J, Ye J R. Research advance in pathogenic
mechanism of pine wood nematode disease ( in Chi-
nese)[J] . Journal of Huazhong Agricultural Universi-
ty (华中农业大学学报), 2003, 22(6): 613 -617.
[8] 　 Tan J J, Ye J R, Wu X Q, et al. A study on disease
development and early diagnosis of pine wood nema-
tode, Bursaphelenchus xylophilus, infection of Japa-
nese black pine[J] . Nematology, 2005, 7(4): 481 -
485.
[9] 　 Zhang X Z, Chen F Y, Wang R F. Experimental tech-
niques of plant physiology ( in Chinese) [M] . Shen-
yang, China, Liaoning Science and Technology Press
(辽宁科学技术出版社), 1994.
[10] Bradford M M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding[J] . Analytical Bio-
chemistry, 1976, 72: 248 -254.
[11] Zou Q. Experimental guide of plant physiobiochemis-
try ( in Chinese) [M] . Beijing: Chinese Agricultural
Press (北京:中国农业出版社), 1995.
[12] Yu F Y, Xu X Z. A review on plant stress physiology
( in Chinese)[ J] . World Forestry Research (世界林
业研究), 2003, 16(5): 6 -11.
[13] Ye J R, Wu X Q. Advances on the physiological and
biochemical aspects of resistance to diseases in trees
( in Chinese)[J] . Forest Research (林业科学研究),
1996, 9(3): 311 -317.
[14] Xu F Y, Ge M H, Zhao Z D, et al. Studies on the re-
lationship of amino acid content of different masson
pine provenances and their resistance to pine wood
nematode (PWN) ( in Chinese)[ J] . Forest Research
(林业科学研究), 1998, 11(3): 313 -318.
[15] Chen Y H, Ye J R, Wei C J, et al. Effects of pine
wood nematode infection on metabolism of active oxy-
gen in Japanese black pine and Slash pine seedlings ( in
Chinese)[ J] . Journal of Nanjing Forestry University
(南京林业大学学报), 2002, 26(4): 19 -22.
责任编辑:李晖
84